Biophysikalische Chemie

Groups

Schematic representation of solar water splitting into O2 and H2 by the enyzmes water-oxidase and hydrogenase.
Schematic representation of solar water splitting into O2 and H2 by the enyzmes wateroxidase and hydrogenase. Shown are the structures of the active sites, particularly the Mn4OxCa cluster of photosystem II in oxygenic photosynthesis and the ‘H-cluster’ of [FeFe]-hydrogenase.1
Figure 1

Department of Biophysical Chemistry

In our department metalloproteins and bioinorganic model complexes are investigated that are related to energy conserving systems. In focus are the water oxidizing enzyme of oxygenic photosynthesis and hydrogenases. We use a variety of different physical techniques to study them, including electrochemistry, X-ray crystallography, magnetic resonance, Mößbauer as well as time resolved optical and vibrational spectroscopy. Particular emphasis is placed on paramagnetic molecules studied by advanced EPR techniques. Most of the investigated systems are prepared in-house, an approach that allows manipulating the samples in an efficient way. The structural information obtained is compared with results from modern quantum chemical calculations aiming at a better understanding of biological function.

In this way insight into enzymatic processes is gained, for example into photosynthetic water splitting and (bio)hydrogen production that can be used for biomimetic chemistry, i.e. to develop catalytic systems in energy research (Fig. 1).

New Methodologies and Instrumentation

EPR instruments available in the Institute: basic technique, frequency band, console, temperature range and magnet (for high field only). Note that two W-band and two pulse Q-band instruments exist with different designs and applications.
EPR instruments available in the Institute: basic technique, frequency band, console, temperature range and magnet (for high field only). Note that two W-band and two pulse Q-band instruments exist with different designs and applications.
Figure 2

One of the aims of our group is to further develop instrumentation and methodology, in particular in the field of multifrequency EPR and related spectroscopies. The special techniques, instruments and frequency bands currently available in our laboratory are summarized in Fig. 2. These span the frequency range from 2 to 244 GHz at fields between 0 and 12 T; for many bands both CW and pulse operation is available. Several instruments allow light access for in situ illumination of samples; for example using a Nd-YAG/OPO laser systems.

During recent years our home-built CW/pulse Q-band ENDOR resonator has been further optimized and a new Q-band pulse bridge with higher microwave pulse power (5-10 W) was set-up (see E. Reijerse). In addition to the newly acquired Bruker W-band EPR instrument a specialized self-constructed W-band spectrometer is available (from FU Berlin, AG Möbius). Their designs are complimentary and open the possibility to measure a large variety of different samples with high sensitivity and spectral resolution using CW/pulse EPR, ENDOR and ELDOR. This includes also the future investigation of small metalloprotein single crystals.

To accomplish crystal growth suitable both for X-ray crystallography and magnetic resonance studies a laboratory exists in our department (see H. Ogata).

Furthermore, the electrochemical techniques used in the past have been extended to include protein film electrochemistry (PFE) under strictly anaerobic conditions, and redox titrations followed either by UV-vis or FTIR in an optical transparent thin layer electrochemical (OTTLE) cell or by EPR (on a series of single samples). Very recently PFE has been combined with surface enhanced IR absorption spectroscopy (SEIRA).

 

DFT and ab initio Calculations

Comparison of spectroscopically obtained and theoretically derived data leading to conclusions about the structure of intermediates and reaction mechanisms.
Comparison of spectroscopically obtained and theoretically derived data leading to conclusions about the structure of intermediates and reaction mechanisms.
Figure 3

DFT and ab initio calculation are performed in our laboratory to verify the measured spectroscopic parameters and obtain reliable electronic and geometrical structures, e. g. of reaction intermediates. The approach is illustrated in Fig. 3. The calculations are performed mostly using the ORCA program package in close collaboration with the department of Prof. Frank Neese (group leaders D. Pantazis and M. van Gastel). The computing facilities at the institute to perform such calculations have been extended.

During the report period the focus has been on calculations of

  • The spin density distribution and hyperfine couplings of triplet states of chlorophylls and carotenoids.2,3
  • Hydrogen bond geometries and their impact on the magnetic resonance parameters of radical ions in photosynthesis.4,5
  • Magnetic resonance properties and structures of manganese complexes.6,7
  • Structure of the water oxidizing complex in PS II.8-10
  • Calculations on the [NiFe]- and [FeFe] hydrogenases and model systems.11,12

Further details are found in the report by F. Neese, D. Pantazis and M. van Gastel.

Hydrogenase

Redox titration of the Fe-S clusters in the [NiFe] Hase of A. aeolicus using EPR samples (see Pandelia et al. PNAS 2011).
Redox titration of the Fe-S clusters in the [NiFe] Hase of A. aeolicus using EPR samples.21
Figure 4 and 5

Structure and function of the enzyme hydrogenase is of central importance for a future biologically based hydrogen production technology and for the design and synthesis of bioinspired model systems for hydrogen conversion catalysis. In our department both the [NiFe] and the [FeFe] hydrogenases are studied in a combined effort by several groups (Reijerse: [FeFe] hydrogenase, Lubitz: [NiFe] hydrogenase, Gärtner: molecular biology and genetics of hydrogenases, Ogata: X-ray crystallography of hydrogenases, Rüdiger: electrochemistry of hydrogenases).

The first crystal structure of a [NiFe] hydrogenase from a photosynthetic bacterium (Allochromatium vinosum) has been obtained to 2.0 Å resolution.13 The structure is similar to that of other known standard hydrogenases from sulfate reducing bacteria except for differences found for the oxidized state Ni-A and the proximal iron-sulfur cluster.

The work on the sulfate-reducing standard hydrogenase Desulfovibrio (D.) vulgaris Miyazaki F, which is also isolated and crystallized in our laboratory, has been continued. In particular the carbon monoxide inhibited states and the light-sensitivity of all intermediates have been measured14 by monitoring the CO/CN-vibrations of the active site via FTIR.

Experiments using surface enhanced infrared absorption (SEIRA) spectroscopy have been successfully performed on this enzyme in cooperation with P. Hildebrandt (TU Berlin)15,16, and first PFE on hydrogenases has also been carried out in our laboratory. Further details of the electronic structure were obtained on hydrogenase samples of D. vulgaris that are labeled with 61Ni [17] or 57Fe.18 Based on these data a reaction scheme for the activation/deactivation of the enzyme, the catalytic cycle, the inhibition by CO and the light sensitivity has been set up.

A new [NiFeSe] hydrogenase has been found in D. vulgaris that is now being studied in detail in our laboratory. Furthermore, the sulfur metabolism of this species has been investigated and two key enzymes, the adenylsulfate reductase and the dissimilatory sulfite reductase have been crystallized (H. Ogata).

A particular problem for employing hydrogenases in biotechnological processes is the oxygen sensitivity of most enzymes. Our group has therefore started, in collaboration with M.-T. Guidici-Orticoni (Marseille), to investigate a [NiFe] hydrogenase from the hyperthermophilic oxygen tolerant bacterium Aquifex (A.) aeolicus.19 By FTIR spectroelectrochemistry in solution four redox intermediate states were characterized; they show significantly higher midpoint potentials than those measured for standard hydrogenases. Carbon monoxide was shown to bind to the active site of this hydrogenase much weaker than to that of D. vulgaris, i.e. the enzyme of A. aeolicus is less susceptible to CO inhibition.20 By EPR redox titrations the types and midpoint potentials of all iron sulfur centers of the electron transfer chain could be determined.21 It was found that the proximal 4Fe-cluster is able to transfer two electrons, which is related to the O2-tolerance of the enzyme (see figure 5).

Based on these results a model for the oxygen tolerance of the hydrogenase I of A. aeolicus was proposed that is based on the unusual electronic properties of the iron sulfur cluster proximal to the catalytic NiFe center. EPR experiments performed on the paramagnetic states of this hydrogenase showed that no Ni-A (oxygen-inhibited state) exists, whereas the enzyme shows signals for the oxidized Ni-B and the reduced Ni-C state. By examining the interaction of the active site with the substrate in the catalytically active Ni-C state using HYSCORE and ENDOR spectroscopy, a weakly bound hydride was found that is lost upon illumination. It is proposed that the strength of the hydride bond is related to the activity of this enzyme.22 A phylogenetic study of aiming at O2 of hydrogenases containing the novel [4Fe-3S]-6Cys cluster was carried out.23

Our work on the [FeFe] hydrogenase using pulse EPR and ENDOR techniques on native and isotope-labeled samples both in sulfate reducing bacteria and in different green algae is described in the report of E. Reijerse. Based on these studies and additional FTIR experiments the complex electronic structure of the hydrogen converting cluster (H cluster) was obtained 24,25 and an important contribution to the mechanism of hydrogen conversion could be made.26,27

The work on the bimetallic hydrogenase enzymes has also been reviewed by our group in several articles.1,28-31

Water Oxidase

ELDOR-detected NMR (top) of the H217O treated PS II with assignment of the 17O resonances in the Mn4O5Ca cluster (bottom). (Rapatskij et al. JACS 2012).
ELDOR-detected NMR (top) of the H217O treated PS II with assignment of the 17O resonances in the Mn4O5Ca cluster (bottom). (Rapatskij et al. JACS 2012).
Figure 6

Light-induced water splitting takes place in photosystem II of all organisms performing oxygenic photosynthesis. The locus of water oxidation is a Mn4OxCa cluster whose exact structure turned out to be difficult to determine by X-ray crystallography due to radiation damage (photo reduction of the Mn ions). Based on structures derived from XAS performed on PS II single crystals, hyperfine and g tensor data from Q-band 55Mn ENDOR/EPR and relaxation data we have developed a model for the electronic structure of the cluster in two states (S0, S2) of the water splitting cycle.32  This includes the spin and oxidation states of the 4 Mn ions as well as the exchange coupling between them and the electronic ground state of the coupled cluster. This model has recently been refined.10 The function of the calcium ion has been probed by replacement of Ca with Sr.33 Furthermore the effect of Ca removal has been examined.34 Many experiments on the water oxidase have been done in collaboration with Johannes Messinger (now at the University of Umea, Sweden).

In all catalytic states (S0 … S4) of the water splitting cycle no Mn2+ has been found. This is, however, expected for the reduced S-states, e.g. the S-2 state that exhibits a well resolved multiline EPR signal. These states can be prepared via chemical reduction using NO, NH2OH, or NH2NH2. For the analysis of the planned experiments on S-2 and for corroborating the conclusions on the other S-states we have studied a series of mixed valence dinuclear manganese complexes together with the group of Karl Wieghardt, including MnIIMnIII systems, using pulse EPR and 55Mn ENDOR spectroscopy at Q-band.35 Recently the binding of water to the Mn4O5Ca cluster has been probed using H217O and detection of the 17O hyperfine and quadrupole couplings by ELDOR-detected NMR (EDNMR). Three different water molecules could be detected and assigned to the cluster (Fig. 6). The substrate water most probably involved in O-O bond formation and oxygen release could also be identified.36 These experiments are essential for understanding the mechanism of water oxidation in PS II.

The data obtained from the various pulse EPR and ENDOR experiments on the water oxidase are important constrains for setting up structural models for the Mn4OxCa cluster including its amino acid surrounding. Together with the department of Frank Neese we have started to explore these possibilities for manganese complexes of different nuclearity and also developed first models for the water oxidase.8,9 The final goal of this project is to fully understand the geometrical and electronic structure of the manganese cluster in its various S-states - including water binding, proton release and dioxygen formation, i. e. to unravel the mechanism of light-induced water splitting in Nature.

Radicals, Radical Pairs and Triplet States in Photosynthesis

(a) Field swept echo-detected EPR (Q-band) of the triplet peridinin in the PCP antenna of A. carterae; (b) Davis ENDOR pulse sequence used; (c) single-crystal-like 1H ENDOR spectrum (Q-band)yielding at least 12 hyperfine couplings including signs; (d) and result of DFT calculation of the spin density distribution (Niklas et al., JACS, 2007).
(a) Field swept echo-detected EPR (Q-band) of the triplet peridinin in the PCP antenna of A. carterae; (b) Davis ENDOR pulse sequence used; (c) single-crystal-like 1H ENDOR spectrum (Q-band)yielding at least 12 hyperfine couplings including signs; (d) and result of DFT calculation of the spin density distribution.2
Figure 7

Time resolved EPR/ENDOR techniques were applied to study short-lived photoexcited states of pigment molecules (chlorophylls, carotenoids). The primary targets were the cofactors in reaction centers of oxygenic photosynthesis. In a different DFG project related species in bacterial photosynthesis have been studied (see report of van Gastel).

Fig. 7 shows the Pulse ENDOR spectrum of the peridinin triplet state in the peridinin-chlorophyll-protein (PCP) antenna of Amphidinium carterae demonstrating for the first time that (in addition to the g and the zero field tensor) the spin density distribution of such a short-lived state (lifetime of ~10 μs) can be obtained via the hyperfine couplings.2 These parameters can also be calculated by DFT methods (see Fig. 7). The analysis of such time resolved experiments gives detailed insight into triplet formation/decay as well as triplet delocalization and triplet transfer, thus allowing a full biophysical characterization of the system. Successful time resolved ENDOR experiment have also been obtained for the triplet states of the primary donors in PS I (3P700) and PS II (3P680), which showed that in both cases the triplet is localized on a monomeric and not on a dimeric chlorophyll species. This contrasts the results obtained for the reaction centers of bacteria (bRC), where a triplet delocalization in the bacteriochlorophyll (BChl) dimer has been found for the primary donors 3P865 and 3P960.37

Further experiments on the unidirectionality of the electron transfer in the bacterial reaction center using various mutants and triplet spectroscopy have been performed 38 and a basic understanding of the spin density distribution of the triplet states of the isolated pigments has been developed based on an orbital mixing model (Marchanka et al., 2009). For further details see the report by Maurice van Gastel.3


Quinone molecules function as electron acceptors in photosynthetic RCs. Their properties are strongly influenced by interactions with the protein environment, in particular by hydrogen bonding. This effect has been studied for the phylloquinone acceptor in PS I where it was shown that a single strong hydrogen bond to this molecule largely determines its structural and functional properties.5 Together with the group of G. Feher (UC San Diego) a detailed study of the strengths and geometries of the hydrogen bonds to the primary quinone acceptor in bacterial reaction centers has been published39 showing the power of Q-band ENDOR on 1H/2H nuclei together with quantum chemical calculations to get insight into these important protein-cofactor interaction. The monoprotonated benzosemiquinone radical, which is an important intermediate in the coupled proton electron transfer in type II reaction centers (bRC, PSII), has recently been studied using EPR/ENDOR techniques.4

In cooperation with the group of K. Möbius (FU Berlin) experiments on the radical pairs created in the charge separation process in bRC were performed using high field dipolar EPR spectroscopy40, from which not only distances but also relative orientations of the radicals can be obtained in the range of ~10 to 40 Å. This enabled a detailed study of light-induced structural changes in the reaction center.41

Ribonucleotide Reductase

Determination of distance and orientation of the two tyrosine radicals in the R2 dimer of mouse RNR via high field PELDOR (Denysenkov et al.; Angew. Chem., 2008).
Determination of distance and orientation of the two tyrosine radicals in the R2 dimer of mouse RNR via high field PELDOR.42
Figure 8

The enzyme ribonucleotide reductase (RNR) produces all four deoxyribonucleotides, the basic building blocks of DNA. They are essential for DNA synthesis and repair in all organisms. RNR is thus under investigation as medical target either for cancer therapy or for a treatment against bacterial/viral infections. RNR stores a stable tyrosyl radical that is required for enzymatic activity and can be destroyed by radical quenchers. Knowledge about the structure of the enzyme and the closer surrounding of the tyrosyl radical might therefore help to design new drugs. We investigated the tertiary structure of the mouse RNRdimer by measuring the distance and relative orientation between the two tyrosyl radicals using high field pulsed electron-electron double resonance (PELDOR) in a cooperation with Dr. M. Bennati (University Frankfurt/Main; MPI for Biophysical Chemistry, Göttingen), see Fig. 8.42

X-ray crystallographic structure of the Mn-RNR (R2F) dimer from C. ammoniagenis at 1.4 Å resolution showing the two Mn ions (green) in each monomer.
X-ray crystallographic structure of the Mn-RNR (R2F) dimer from C. ammoniagenis at 1.4 Å resolution showing the two Mn ions (green) in each monomer.44
Figure 9

Recently an RNR has been described from Chlamydia that contains an FeMn complex, instead of a dinuclear iron cluster, that generates the tyrosyl radical in RNR. Interestingly, a tyrosine radical is not present in this RNR. We have studied together with the group of G. Auling (Hannover) the RNR of Corynebacterium ammoniagenes and found a MnMn complex in this species by X-ray crystallography (Fig. 9).43 The analysis of the complex manganese EPR spectra, in which also a tyrosine radical is involved, has also been accomplished.44

(a) Ribbon diagram of the E. coli bacterioferritin homodimer with two identical subunits each housing a dimetal center (2 Mn ions)and a Zn-chlorin at the interface; (b) estimated distances between the Mn2, tyrosine and ZnCe6 compared to (c) the arrangement in the PS II reaction center (Conlan et.al., BBA 2009).
(a) Ribbon diagram of the E. coli bacterioferritin homodimer with two identical subunits each housing a dimetal center (2 Mn ions)and a Zn-chlorin at the interface; (b) estimated distances between the Mn2, tyrosine and ZnCe6 compared to (c) the arrangement in the PS II reaction center.50
Figure 10

Protein Models

De novo synthesis of proteins has been pursued in our group to create small systems (“maquettes”) for study in protein-cofactor interactions and to learn about the minimal requirements to obtain functional systems (e. g. for electron transfer or catalysis). This project is carried out in cooperation with Prof. W. Gärtner. The focus has recently been on iron-sulfur clusters. It could be shown that clusters spontaneously form in solution with small peptides.45 The Fe4S4 centers FA and FB in PS I were modelled and characterized with various techniques; they showed unusually low redox potentials.46 In cooperation with D. Noy (Weizmann Institute, Rehovot, Israel) Fe4S4 clusters were synthesized and characterized embedded in a novel non-natural α-helical coiled coil protein fold.47 In a different approach attempts have been made to determine the pathway of Fe3S4 cluster formation and to distinguish it from that of Fe4S4.48 Furthermore bacteriochlorophyll dimer formation has been studied in de novo designed peptides.49

In another project related to “artificial photosynthesis” we have participated in characterizing an engineered reaction center based on modified bacterioferritin that contains a light-excitable Zn-chlorin and a dinuclear manganese cluster coupled by a tyrosine residue in cooperation with T. Wydrzynski, Canberra.50 It could be shown that photocatalytic oxidation of the Mn site occurs, most probably via the redox active tyrosine (see Fig. 10).


References

        1) Lubitz, W.; Reijerse, E. J.; Messinger, J. Solar Water-Splitting into H2 and O2: Design Principles of Photosystem II and Hydrogenases. Energy Environ. Sci. 2008, 1, 15-31.

        (2)     Niklas, J.; Schulte, T.; Prakash, S.; van Gastel, M.; Hofmann, E.; Lubitz, W. Spin-Density Distribution of the Carotenoid Triplet State in the Peridinin-Chlorophyll-Protein Antenna. A Q-Band Pulse Electron-Nuclear Double Resonance and Density Functional Theory Study. J. Am. Chem. Soc. 2007, 129, 15442-15443.

        (3)     Marchanka, A.; Lubitz, W.; van Gastel, M. Spin Density Distribution of the Excited Triplet State of Bacteriochlorophylls. Pulsed ENDOR and DFT Studies. J. Phys. Chem. B 2009, 113, 6917-6927.

        (4)     Flores, M.; Okamura, M. Y.; Niklas, J.; Pandelia, M. E.; Lubitz, W. Pulse Q-Band EPR and ENDOR Spectroscopies of the Photochemically Generated Monoprotonated Benzosemiquinone Radical in Frozen Alcoholic Solution. J. Phys. Chem. B 2012, 116, 8890-8900.

        (5)     Niklas, J.; Epel, B.; Antonkine, M. L.; Sinnecker, S.; Pandelia, M. E.; Lubitz, W. Electronic Structure of the Quinone Radical Anion A1·- of Photosystem I Investigated by Advanced Pulse EPR and ENDOR Techniques. J. Phys. Chem. B 2009, 113, 10367-10379.

        (6)     Zein, S.; Kulik, L. V.; Yano, J.; Kern, J.; Pushkar, Y.; Zouni, A.; Yachandra, V. K.; Lubitz, W.; Neese, F.; Messinger, J. Focusing the View on Nature's Water-Splitting Catalyst. Phil. Trans. R. Soc. 2008, 363, 1167-1177.

        (7)     Pantazis, D. A.; Orio, M.; Petrenko, T.; Zein, S.; Bill, E.; Lubitz, W.; Messinger, J.; Neese, F. A New Quantum Chemical Approach to the Magnetic Properties of Oligonuclear Transition-Metal Complexes: Application to a Model for the Tetranuclear Manganese Cluster of Photosystem II. Chem. Eur. J. 2009, 15, 5108-5123.

        (8)     Pantazis, D. A.; Orio, M.; Petrenko, T.; Zein, S.; Lubitz, W.; Messinger, J.; Neese, F. Structure of the Oxygen-Evolving Complex of Photosystem II: Information on the S2 State Through Quantum Chemical Calculation of Its Magnetic Properties. Phys. Chem. Chem. Phys. 2009, 11, 6788-6798.

        (9)     Pantazis, D. A.; Ames, W.; Cox, N.; Lubitz, W.; Neese, F. Two Interconvertible Structures That Explain the Spectroscopic Properties of the Oxygen-Evolving Complex of Photosystem II in the S2 State. Angew. Chem. Int. Ed. 2012, 51, 9935-9940.

     (10)     Ames, W.; Pantazis, D. A.; Krewald, V.; Cox, N.; Messinger, J.; Lubitz, W.; Neese, F. Theoretical Evaluation of Structural Models of the S2 State in the Oxygen Evolving Complex of Photosystem II: Protonation States and Magnetic Interactions. J. Am. Chem. Soc. 2011, 133, 19743-19757.

     (11)     Kampa, M.; Lubitz, W.; van Gastel, M.; Neese, F. Computational Study of the Electronic Structure and Magnetic Properties of the Ni-C State in [NiFe] Hydrogenases Including the Second Coordination Sphere. J. Biol. Inorg. Chem 2012, accepted for publication.

     (12)     Silakov, A.; Olsen, M. T.; Sproules, S.; Reijerse, E. J.; Rauchfuss, T. B.; Lubitz, W. EPR/ENDOR, Mössbauer, and Quantum-Chemical Investigations of Diiron Complexes Mimicking the Active Oxidized State of [FeFe]Hydrogenase. Inorg. Chem. 2012, 51, 8617-8628.

     (13)     Ogata, H.; Kellers, P.; Lubitz, W. The Crystal Structure of the [NiFe] Hydrogenase From the Photosynthetic Bacterium Allochromatium Vinosum: Characterization of the Oxidized Enzyme (Ni-A State). J. Mol. Biol. 2010, 402, 428-444.

     (14)     Pandelia, M. E.; Ogata, H.; Currell, L. J.; Flores, M.; Lubitz, W. Probing Intermediates in the Activation Cycle of [NiFe] Hydrogenase by Infrared Spectroscopy: The Ni-SIr State and Its Light Sensitivity. J. Biol. Inorg. Chem. 2009, 14, 1227-1241.

     (15)     Millo, D.; Pandelia, M. E.; Utesch, T.; Wisitruangsakul, N.; Mroginski, M. A.; Lubitz, W.; Hildebrandt, P.; Zebger, I. Spectroelectrochemical Study of the [NiFe] Hydrogenase From Desulfovibrio Vulgaris Miyazaki F in Solution and Immobilized on Biocompatible Gold Surfaces. J. Phys. Chem. B 2009, 113, 15344-15351.

     (16)     Millo, D.; Hildebrandt, P.; Pandelia, M. E.; Lubitz, W.; Zebger, I. SEIRA Spectroscopy of the Electrochemical Activation of an Immobilized [NiFe] Hydrogenase Under Turnover and Non-Turnover Conditions. Angew. Chem. Int. Ed. 2011, 50, 2632-2634.

     (17)     Flores, M.; Agrawal, A. G.; van Gastel, M.; Gärtner, W.; Lubitz, W. Electron-Electron Double Resonance-Detected NMR to Measure Metal Hyperfine Interactions: 61Ni in the Ni-B State of the [NiFe] Hydrogenase of Desulfovibrio Vulgaris Miyazaki F. J. Am. Chem. Soc. 2008, 130, 2402-2403.

     (18)     Pandelia, M. E.; Ogata, H.; Lubitz, W. Intermediates in the Catalytic Cycle of [NiFe] Hydrogenase: Functional Spectroscopy of the Active Site. Chem. Phys. Chem. 2010, 11, 1127-1140.

     (19)     Pandelia, M. E.; Fourmond, V.; Tron-Infossi, P.; Lojou, E.; Bertrand, P.; Léger, C.; Giudici-Orticoni, M. T.; Lubitz, W. Membrane-Bound Hydrogenase I From the Hyperthermophilic Bacterium Aquifex Aeolicus: Enzyme Activation, Redox Intermediates and Oxygen Tolerance. J. Am. Chem. Soc. 2010, 132, 6991-7004.

     (20)     Pandelia, M. E.; Infossi, P.; Giudici-Orticoni, M. T.; Lubitz, W. The Oxygen-Tolerant Hydrogenase I From Aquifex Aeolicus Weakly Interacts With Carbon Monoxide: An Electrochemical and Time-Resolved FTIR Study. Biochemistry 2010, 49, 8873-8881.

     (21)     Pandelia, M. E.; Nitschke, W.; Infossi, P.; Giudici-Orticoni, M. T.; Bill, E.; Lubitz, W. Characterization of a Unique [FeS] Cluster in the Electron Transfer Chain of the Oxygen Tolerant [NiFe] Hydrogenase From Aquifex Aeolicus. P. Natl. Acad. Sci. USA 2011, 108, 6097-6102.

     (22)     Pandelia, M. E.; Infossi, P.; Stein, M.; Giudici-Orticoni, M. T.; Lubitz, W. Spectroscopic Characterization of the Key Catalytic Intermediate Ni-C in the O2-Tolerant [NiFe] Hydrogenase I From Aquifex Aeolicus: Evidence of a Weakly Bound Hydride. Chem. Commun. 2012, 48, 823-825.

     (23)     Pandelia, M. E.; Lubitz, W.; Nitschke, W. Evolution and Diversification of Group 1 [NiFe] Hydrogenases. Is There a Phylogenetic Marker for O2-Tolerance? Biochimica et Biophysica Acta (BBA) - Bioenergetics 2012, 1817, 1565-1575.

     (24)     Silakov, A.; Reijerse, E. J.; Albracht, S. P. J.; Hatchikian, E. C.; Lubitz, W. The Electronic Structure of the H-Cluster in the [FeFe]-Hydrogenase From Desulfovibrio Desulfuricans: A Q-Band 57Fe ENDOR and HYSCORE Study. J. Am. Chem. Soc. 2007, 129, 11447-11458.

     (25)     Kamp, C.; Silakov, A.; Winkler, M.; Reijerse, E. J.; Lubitz, W.; Happe, T. Isolation and First EPR Characterization of the [FeFe]-Hydrogenases From Green Algae. Biochim. Biophys. Acta 2008, 1777, 410-416.

     (26)     Silakov, A.; Wenk, B.; Reijerse, E.; Lubitz, W. 14N HYSCORE Investigation of the H-Cluster of [FeFe] Hydrogenase: Evidence for a Nitrogen in the Dithiol Bridge. Phys. Chem. Chem. Phys. 2009, 11, 6592-6599.

     (27)     Adamska, A.; Silakov, A.; Lambertz, C.; Rüdiger, O.; Happe, T.; Reijerse, E.; Lubitz, W. Identification and Characterization of the 'Super-Reduced' State of the H-Cluster in [FeFe] Hydrogenase: A New Building Block for the Catalytic Cycle? Angew. Chem. Int. Ed. 2012, 51, 11458-11462.

     (28)     Lubitz, W.; Reijerse, E.; van Gastel, M. [NiFe] and [FeFe] Hydrogenases Studied by Advanced Magnetic Resonance Techniques. Chem. Rev. 2007, 107, 4331-4365.

     (29)     van Gastel, M.; Lubitz, W. EPR Investigation of [NiFe] Hydrogenases. In High Resolution EPR: Applications to Metalloenzymes and Metals in Medicine; Hanson, G. R., Berliner, L. J., Eds.; 2009.

     (30)     Ogata, H.; Lubitz, W.; Higuchi, Y. [NiFe] Hydrogenases: Structural and Spectroscopic Studies of the Reaction Mechanism. Dalton Trans 2009,  7577-7587.

     (31)     Shafaat, H. S.; Rüdiger, O.; Ogata, H.; Lubitz, W. [NiFe] Hydrogenases: A Common Active Site for Function in Diverse Environments. Biochim. Biophys. Acta 2012, accepted for publication.

     (32)     Kulik, L. V.; Epel, B.; Lubitz, W.; Messinger, J. Electronic Structure of the Mn4OxCa Cluster in the S0 and S2 States of the Oxygen-Evolving Complex of Photosystem II Based on Pulse 55Mn ENDOR and EPR Spectroscopy. J. Am. Chem. Soc. 2007, 129, 13421-13435.

     (33)     Cox, N.; Rapatskiy, L.; Su, J. H.; Pantazis, D. A.; Sugiura, M.; Kulik, L. V.; Dorlet, P.; Rutherford, A. W.; Neese, F.; Boussac, A.; Lubitz, W.; Messinger, J. Effect of Ca2+/Sr2+ Substitution on the Electronic Structure of the Oxygen-Evolving Complex of Photosystem II: A Combined Multifrequency EPR, 55Mn-ENDOR, and DFT Study of the S2 State. J. Am. Chem. Soc. 2011, 133, 3635-3648.

     (34)     Lohmiller, T.; Cox, N.; Su, J. H.; Messinger, J.; Lubitz, W. The Basic Properties of the Electronic Structure of the Oxygen-Evolving Complex of Photosystem II Are Not Perturbed by Ca2+ Removal. J. Biol. Chem. 2012, 287, 24721-24733.

     (35)     Cox, N.; Ames, W.; Epel, B.; Kulik, L. V.; Rapatskiy, L.; Neese, F.; Messinger, J.; Wieghardt, K.; Lubitz, W. Electronic Structure of a Weakly Antiferromagnetically Coupled MnIIMnIII Model Relevant to Manganese Proteins: A Combined EPR, 55Mn-ENDOR, and DFT Study. Inorg. Chem. 2011, 50, 8238-8251.

     (36)     Rapatskiy, L.; Cox, N.; Savitsky, A.; Ames, W.; Sander, J.; Nowaczyk, M.; Rögner, M.; Boussac, A.; Neese, F.; Messinger, J.; Lubitz, W. Detection of the Water Binding Sites of the Oxygen-Evolving Complex of Photosystem II Using W-Band 17O ELDOR-Detected NMR Spectroscopy. J. Am. Chem. Soc. 2012, 134, 16619-16634.

     (37)     Marchanka, A.; Lubitz, W.; van Gastel, M.; Plato, M. Comparative ENDOR Study at 34 GHz and the Triplet State of the Primary Donor in Bacterial Reaction Centers of Rb. Sphaeroides and Bl. Viridis. Photosynth. Res. 2012, in press.

     (38)     Marchanka, A.; Paddock, M.; Lubitz, W.; van Gastel, M. Low-Temperature Pulsed EPR Study at 34 GHz of the Triplet States of the Primary Electron Donor P865 and the Carotenoid in Native and Mutant Bacterial Reaction Centers of Rhodobacter Sphaeroides. Biochemistry 2007, 46, 14782-14794.

     (39)     Flores, M.; Isaacson, R.; Abresch, E.; Calvo, R.; Lubitz, W.; Feher, G. Protein-Cofactor Interactions in Bacterial Reaction Centers From Rhodobacter Sphaeroides R-26: II. Geometry of the Hydrogen Bonds to the Primary Quinone QA·- by 1H and 2H ENDOR Spectroscopy. Biophys. J. 2007, 92, 671-682.

     (40)     Savitsky, A.; Dubinskii, A. A.; Flores, M.; Lubitz, W.; Möbius, K. Orientation-Resolving Pulsed Electron Dipolar High-Field EPR Spectroscopy on Disordered Solids: I. Structure of Spin-Correlated Radical Pairs in Bacterial Photosynthetic Reaction Centers. J. Phys. Chem. B 2007, 111, 6245-6262.

     (41)     Flores, M.; Savitsky, A.; Paddock, M. L.; Abresch, E. C.; Dubinskii, A. A.; Okamura, M. Y.; Lubitz, W.; Möbius, K. Electron-Nuclear and Electron-Electron Double Resonance Spectroscopies Show That the Primary Quinone Acceptor QA in Reaction Centers From Photosynthetic Bacteria Rhodobacter Sphaeroides Remains in the Same Orientation Upon Light-Induced Reduction. J. Phys. Chem. B 2010, 114, 16894-16901.

     (42)     Denysenkov, V. P.; Biglino, D.; Lubitz, W.; Prisner, T. F.; Bennati, M. Structure of the Tyrosyl Biradical in Mouse R2 Ribonucleotide Reductase From High-Field PELDOR. Angew. Chem. Int. Ed. 2008, 47, 1224-1227.

     (43)     Ogata, H.; Stolle, P.; Stehr, M.; Auling, G.; Lubitz, W. Crystallization and Preliminary X-Ray Analysis of the Small Subunit (R2F) of Native Ribonucleotide Reductase From Corynebacterium Ammoniagenes. Acta Crystallographica Section F-Structural Biology and Crystallization Communications 2009, 65, 878-880.

     (44)     Cox, N.; Ogata, H.; Stolle, P.; Reijerse, E.; Auling, G.; Lubitz, W. A Tyrosyl-Dimanganese Coupled Spin System Is the Native Metalloradical Cofactor of the R2F Subunit of the Ribonucleotide Reductase of Corynebacterium Ammoniagenes. J. Am. Chem. Soc. 2010, 132, 11197-11213.

     (45)     Koay, M. S.; Antonkine, M. L.; Gärtner, W.; Lubitz, W. Modelling Low-Potential [Fe4S4] Clusters in Proteins. Chem. Biodivers. 2008, 5, 1571-1587.

     (46)     Antonkine, M. L.; Koay, M. S.; Epel, B.; Breitenstein, C.; Gopta, O.; Gärtner, W.; Bill, E.; Lubitz, W. Synthesis and Characterization of De Novo Designed Peptides Modelling the Binding Sites of [4Fe-4S] Clusters in Photosystem I. Biochim. Biophys. Acta, Bioenerg. 2009, 1787, 995-1008.

     (47)     Grzyb, J.; Xu, F.; Nanda, V.; Luczkowska, R.; Reijerse, E.; Lubitz, W.; Noy, D. Empirical and Computational Design of Iron-Sulfur Cluster Proteins. Biochimica et Biophysica Acta (BBA) - Bioenergetics 2012, 1817, 1256-1262.

     (48)     Hoppe, A.; Pandelia, M. E.; Gärtner, W.; Lubitz, W. [Fe4S4]- and [Fe3S4]-Cluster Formation in Synthetic Peptides. Biochimica et Biophysica Acta (BBA) - Bioenergetics 2011, 1807, 1414-1422.

     (49)     Cohen-Ofri, I.; van Gastel, M.; Grzyb, J.; Brandis, A.; Pinkas, I.; Lubitz, W.; Noy, D. Zinc-Bacteriochlorophyllide Dimers in De Novo Designed Four-Helix Bundle Proteins. A Model System for Natural Light Energy Harvesting and Dissipation. J. Am. Chem. Soc. 2011, 9526-9535.

(50)      Conlan, B.; Cox, N.; Su, J. H.; Hillier, W.; Messinger, J.; Lubitz, W.; Dutton, P. L.; Wydrzynski, T. Photo-Catalytic Oxidation of a Di-Nuclear Manganese Centre in an Engineered Bacterioferritin 'Reaction Centre'. Biochim. Biophys. Acta, Bioenerg. 2009, 1787, 1112-1121.

(c) MPI für Chemische Energiekonversion, Mülheim a.d.R. 2012